The subject invention is in the field of sustained drug delivery using micro-encapsulation of bioactive agents. In particular, the invention describes improved methods for incorporating polyanionic bioactive agents into polymeric microspheres and/or nanospheres through the use of a condensing agent, as well as microspheres and/or nanospheres prepared by the method.
Gene therapy was originally conceived as a specific gene replacement therapy for correcting heritable defects by delivering functionally active therapeutic genes into targeted cells. Initial efforts toward somatic gene therapy have largely relied on indirect means of introducing genes into tissues, called ex vivo gene therapy. In ex vivo protocols, target cells are removed from the body, transfected or infected in vitro with vectors carrying recombinant genes, and re-implanted into the body (xe2x80x9cautologous cell transferxe2x80x9d). A variety of transfection techniques are currently available and used to transfer DNA in vitro into cells, including calcium phosphate-DNA precipitation, DEAE-Dextran transfection, electroporation, liposome mediated DNA transfer, and transduction with recombinant viral vectors. Such ex vivo treatment protocols have been proposed to transfer DNA into a variety of different cell types including epithelial cells (Morgan et al., U.S. Pat. No. 4,868,116; Morgan and Mulligan, WO87/00201; Morgan et al., 1987, Science 237:1476-1479; Morgan and Mulligan, U.S. Pat. No. 4,980,286), endothelial cells (WO89/05345), hepatocytes (Wilson and Mulligan, WO89/07136; Wolff et al., 1987, Proc. Natl. Acad. Sci. USA 84:3344-3348; Ledley et al., 1987, Proc. Natl. Acad. Sci. USA 84:5335-5339; Wilson et al., 1990, Proc. Natl. Acad. Sci. USA 87:8437-8441), fibroblasts (Palmer et al., 1987, Proc. Natl. Acad. Sci. USA 84:1055-1059; Anson et al., 1987, Mol. Biol. Med. 4:11-20; Rosenberg et al., 1988, Science 242:1575-1578; Naughton and Naughton, U.S. Pat. No. 4,963,489), lymphocytes (Anderson et al., U.S. Pat. No. 5,399,346; Blaese et al., 1995, Science 270:475-480) and hematopoietic stem cells (Lim et al., 1989, Proc. Natl. Acad. Sci. USA 86:8892-8896; Anderson et al., U.S. Pat. No. 5,399,346).
Direct in vivo gene transfer has recently been attempted with formulations of DNA trapped in liposomes (Ledley et al., 1987, J. Pediatrics 110:1), in proteoliposomes that contain viral envelope receptor proteins (Nicolau et al., 1983, Proc. Natl. Acad. Sci. USA 80:1068), and with DNA coupled to a polylysine-glycoprotein carrier complex. In addition, xe2x80x9cgene gunsxe2x80x9d have been used for gene delivery into cells (Australian Patent No. 9068389). Some have even speculated that naked DNA, or DNA associated with liposomes, can be formulated in liquid carrier solutions suitable for injection into interstitial spaces for transfer of DNA into cells (Felgner, WO90/11092).
Perhaps one of the greatest problems associated with currently devised gene therapies, whether ex vivo or in vivo, is the inability to transfer DNA efficiently into a targeted cell population and to achieve high level expression of the gene product in vivo. Viral vectors are regarded as the most efficient system, and recombinant, replication-defective viral vectors have been used to transduce (via infection) cells both ex vivo and in vivo. Such vectors have included retroviral, adenoviral and adeno-associated, and herpes viral vectors. While highly efficient at gene transfer, the major disadvantages associated with the use of viral vectors include the inability of many viral vectors to infect non-dividing cells; problems associated with insertional mutagenesis; inflammatory reactions to the virus and potential helper virus production; antibody responses to the viral coats; and the potential for production and transmission of harmful virus to other human patients.
The efficiency of gene transfer into cells directly influences the resultant gene expression levels. In addition to the general low efficiency with which most cell types take up and express foreign DNA, many targeted cell populations are found in very low numbers in the body, so that the low efficiency of presentation of DNA to the specific targeted cell types further diminished the overall efficiency of gene transfer.
In many approaches aimed at increasing the efficiency of gene transfer into cells, the nucleic acid is typically complexed with carriers that facilitate the transfer of the DNA across the cell membrane for delivery to the nucleus. The carrier molecules bind and condense DNA into small particles which facilitate DNA entry into cells through endocytosis or pinocytosis. In addition, the carrier molecules act as scaffolds to which ligands may be attached in order to achieve site- or cell-specific targeting of DNA.
The most common DNA condensing agents used in the development of nonviral gene delivery systems include polylysine (Laemmli, 1975, Proc. Natl. Acad. Sci. USA 72:4288-92; Wolfert and Seymour, 1996, Gene Therapy 3:269-73) and low molecular weight glycopeptides (Wadhwa et al., 1995, Bioconjugate Chemistry 6:283-291). Polylysine amino groups have been derivatized with transferrin, glycoconjugates, folate, lectins, antibodies, or other proteins to provide specificity in cell recognition, without compromising the polylysine""s binding affinity for DNA.
Clearly, improved methods of gene delivery are needed. Such methods should be amenable to use with virtually any gene of interest and should permit the introduction of genetic material into a variety of cells and tissues.
Receptor-mediated gene delivery has emerged as a potentially useful approach for introduction of DNA into cells in vivo. An advantage of this gene delivery method is the ability to target DNA to specific tissue or cell types based on the recognition of ligands by unique receptors expressed on the cell surface (Wu et al., 1988, J. Biol. Chem. 263:14621-14624; Christiano et al., 1993, Proc. Natl. Acad. Sci. USA 90:2122-2126; Huckett et al., 1990, Biochem. Phannacol. 40:253-263; Perales et al., 1994, Eur. J. Biochem. 226:255-266). In addition, this particular delivery system is not limited by the size of the DNA and the system does not involve the use of infectious agents.
Receptor-mediated gene transfer has considerable potential for use in human gene therapy if the method can be developed to a point where it is both a reliable and efficient approach for delivery in targeted host cells. The major shortcomings of currently available techniques are transient, variable and low level expression of the transferred DNA. Any method designed to increase the efficiency of transfer of DNA into the cell will facilitate the successful development of receptor-mediated gene delivery protocols.
Oftentimes, it is desirable to deliver pharmaceutical or other bioactive agents intracellularly rather than, or in addition to, extracellularly. Such applications are particularly useful where, for example, the bioactive agent cannot easily penetrate or traverse the cellular membrane. Examples of such bioactive agents include oligonucleotides such as antisense DNA and RNA, ribozymes, DNA for gene therapy, transcription factors, growth factor binding proteins, signalling receptors and the like. Also desirable is sustaining this delivery of bioactive agents over an extended period of time.
Microspheres and/or nanospheres are a widely used vehicle for delivering drugs intracellularly, and for sustaining the delivery for an extended time. Generally, microspheres and/or nanospheres comprise a biocompatible biodegradable polymeric core having a bioactive agent incorporated therein. Microspheres are typically spherical and have an average diameter of about 1 to 900 xcexcm, while nanospheres are typically spherical and have an average diameter of less than 1 xcexcm, usually less than about 300 nm.
Advantages of microsphere and/or nanosphere (hereinafter collectively xe2x80x9cmicrospherexe2x80x9d) bioactive formulations include their ability to enter cells and penetrate intracellular junctions. Another advantage of microspheres is their ability to provide sustained or controlled release of bioactive agents. Thus, microspheres provide a means for intracellular as well as extracellular controlled or sustained delivery of pharmaceutical and other bioactive agents.
Often, the pharmaceutical or bioactive agent is a polyionic molecule. These polyionic molecules often do not pack well into microspheres and thus have reduced incorporation efficiencies. Examples of polyanionic bioactive agents include nucleic acids, for example DNA and RNA; many proteins, for example bone acidic glycoprotein 75,000 da (BAG75) and other phosphoproteins of bones and teeth, xcex2-lactoglobulin, and phycocyanin; and glycosaminoglycans, for example heparin, heparan sulfate, chondroitin sulfate, polyuronic acid, and hyaluronic acid. In particular, DNA is a highly desirable polyanionic bioactive agent for delivery via microspheres.
However, efforts to formulate DNA within microspheres have been hampered by several difficulties. For example, present methods exhibit very low efficiencies of incorporationxe2x80x94most of the DNA present in the emulsion used to prepare the microspheres does not get into the microspheres. Methods that enhance the efficiency of DNA incorporation would have the beneficial effect of requiring less starting DNA to produce an end product with a given amount of incorporated DNA. Such methods might also increase the amount of DNA incorporated into each microsphere, allowing the introduction of fewer microspheres into the treatment site to deliver a given amount of total DNA to a patient.
Moreover, incorporation of DNA into microspheres is plagued by fragmentation of the DNA. In one common method, DNA microspheres are formed using a water-in-oil-in-water double emulsion method. Unfortunately, each of the two emulsifying steps frequently involves sonication, which causes fragmentation of the DNA.
Having available methods for increasing the efficiency of incorporating DNA within microspheres without inducing DNA fragmentation would be extremely advantageous. Such DNA-containing microspheres would facilitate intracellular as well as extracellular controlled or sustained release of therapeutic DNA agents at the site of medical intervention. These microspheres would be particularly advantageous for delivering DNA for use in gene therapy.
An alternative method for intracellular delivery is liposomes. However, liposomes, including polycationic liposomes, do not have the desirable sustained release properties that microspheres exhibit, as they tend to be less stable and to release their contents rapidly. Thus, for many purposes, lipsomal delivery systems are not as effective as microsphere delivery systems. Furthermore, liposomes are made using completely different methods from those used to make microspheres.
Many advantages are provided by the present invention, which in one aspect is directed to a method of making microspheres containing polyanionic bioactive agents. The efficiency of incorporation of the polyanionic bioactive agents into the microspheres is increased by using a condensing agent to condense the polyanionic bioactive agent during the manufacture of the microsphere.
In its broadest sense, the method involves the use of a condensing agent in one of the phases used to produce the microsphere. Many methods for making microspheres, which are known in the art, are amenable to the use of the condensing agent as described herein. These methods include, but are not limited to, water-in-oil-in-water (W/O/W) double emulsions, water-in-oil (W/O) or oil-in-water (O/W) single emulsions, salting out, diafiltration, coacervation, hot melt, and spray drying. A preferred method for use in conjunction with a condensing agent is the water-in-oil-in-water double emulsion method.
In one illustrative embodiment of the invention, microspheres containing polyanionic bioactive agents are prepared using a water-in-oil-in-water double emulsion method, which method comprises the steps of: (a) dissolving at least one polymer in a water-immiscible organic solvent to yield an organic phase; (b) dissolving a polyanionic bioactive agent in aqueous solution to yield a first aqueous phase; (c) emulsifying the organic and first aqueous phases to yield a first milky emulsion; (d) dissolving a condensing agent in aqueous solution to yield a second aqueous phase; (e) emulsifying the first milky emulsion and the second aqueous phase to yield a second milky emulsion; and (f) removing the organic solvent from the second milky emulsion to yield microspheres containing condensed polyanionic bioactive agent. The removal of the organic solvent in the final step is preferably by means of evaporation.
In a preferred embodiment, the polymer is a biocompatible biodegradable polymer, such as polylactic polyglycolic acid (PLGA). Preferred water-immiscible organic solvents include chloroform and methylene dichloride. In an alternative preferred embodiment, the second aqueous phase may optionally include an emulsifying agent; this emulsifying agent is preferably polyvinyl alcohol (PVA).
Many polyanionic bioactive agents are useful in the present invention, including nucleic acids, such as DNA, RNA, or oligonucleotides; proteins, such as bone acidic glycoprotein 75,000 da (BAG75) and other phosphoproteins of bones and teeth, and xcex2-lactoglobulin; and glycosaminoglycans, such as polyuronic acid and hyaluronic acid. In a preferred embodiment, the polyanionic bioactive agent is a nucleic acid, particularly DNA. For the condensing agent, a preferred embodiment is a polycation, preferably polylysine, especially poly-L-lysine, and derivatives thereof. Alternatively, the polycation may also preferably be a polypeptide, for example myelin basic protein, histones, DNA binding domains from histones, DNA binding domains from, for example, transcription factors, and synthetic polypeptides made up of one or more of these domains.
In another aspect, the present invention is directed to microspheres containing polyanionic bioactive agents. Generally, the microspheres comprise a polymeric core, preferably a biocompatible biodegradable polymeric core, and have entrapped, entrained, embedded, encapsulated, or otherwise incorporated therein a condensed polyanionic bioactive agent. Thus, in one illustrative embodiment, the microspheres comprise a polymer, preferably a biocompatible biodegradable polymer, a condensing agent, and a polyanionic bioactive agent. In preferred embodiments, the polyanionic bioactive agent is a nucleic acid, particularly DNA, as described above. Similarly, as described above, preferred condensing agents are polycations, particularly polylysine and derivatives thereof. A preferred biocompatible biodegradable polymer is polylactic polyglycolic acid.